Method of sterilization in diffusion restricted environments

ABSTRACT

Methods for peroxide vapor sterilization of medical devices and similar instruments having long narrow lumens or diffusion restricted areas includes the step of contacting the article to be sterilized or a diffusion-restricted environment containing said article with a source of peroxide prior to exposure to a vacuum or a vacuum followed by plasma. The methods are such that, upon vaporization of the source of peroxide caused by the vacuum, the peroxide remains in contact with the article for a time sufficient to achieve sterilization.

This application is a continuation-in-part of application Ser. No.08/628,965, filed Apr. 4, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for using a source of peroxide andnegative pressure to sterilize articles such as medical instruments, andmore particularly, to methods which include the step of contacting thearticles or the diffusion-restricted environment containing the articleswith a source of peroxide prior to exposure to negative pressure ornegative pressure combined with plasma.

2. Description of the Related Art

Medical instruments have traditionally been sterilized using eitherheat, such as is provided by steam, or a chemical, such as formaldehydeor ethylene oxide in the gas or vapor state. Each of these methods hasdrawbacks. Many medical devices, such as fiberoptic devices, endoscopes,power tools, etc. are sensitive to heat, moisture, or both. Formaldehydeand ethylene oxide are both toxic gases that pose a potential hazard tohealthcare workers. Problems with ethylene oxide are particularlysevere, because its use requires long aeration times to remove the gasfrom articles that have been sterilized. This makes the sterilizationcycle time undesirably long.

Sterilization using liquid hydrogen peroxide solution has been found torequire high concentration of sterilant, extended exposure time and/orelevated temperatures. However, sterilization using hydrogen peroxidevapor has been shown to have some advantages over other chemicalsterilization processes (see, e.g., U.S. Pat. Nos. 4,169,123 and4,169,124). The combination of hydrogen peroxide with a plasma providescertain additional advantages, as disclosed in U.S. Pat. No. 4,643,876,issued Feb. 17, 1987 to Jacobs et al.. U.S. Pat. No. 4,756,882, issuedJul. 12, 1988 also to Jacobs et al. discloses the use of hydrogenperoxide vapor, generated from an aqueous solution of hydrogen peroxide,as a precursor of the reactive species generated by a plasma generator.The combination of hydrogen peroxide vapor diffusing into closeproximity with the article to be sterilized and plasma acts to sterilizethe articles, even within closed packages. Further, these methods ofcombining hydrogen peroxidevapor with a plasma, while useful in "open"systems, have been found to be inadequate to effect sterilization inarticles having diffusion-restricted areas, since the methods aredependent upon diffusion of the sterilant vapor into close proximitywith the article before sterilization can be achieved. Thus, thesemethods have been found to require high concentration of sterilant,extended exposure time and/or elevated temperatures when used on long,narrow lumens. For example, lumens longer than 27 cm and/or having aninternal diameter of less than 0.3 cm have been particularly difficultto sterilize. Thus, no simple, safe, effective method of sterilizingsmaller lumens exists in the prior art.

The sterilization of articles containing diffusion-restricted areas,such as long narrow lumens, therefore presents a special challenge.Methods that use hydrogen peroxide vapor that has been generated from anaqueous solution of hydrogen peroxide have certain disadvantages,because:

1. Water has a higher vapor pressure than hydrogen peroxide and willvaporize faster than hydrogen peroxide from an aqueous solution.

2. Water has a lower molecular weight than hydrogen peroxide and willdiffuse faster than hydrogen peroxide in the vapor state.

Because of this, when an aqueous solution of hydrogen peroxide isvaporized in the area surrounding the items to be sterilized, the waterreaches the items first and in higher concentration. The water vaportherefore becomes a barrier to the penetration of hydrogen peroxidevapor into diffusion restricted areas, such as small crevices and longnarrow lumens. One cannot solve the problem by removing water from theaqueous solution and using more concentrated hydrogen peroxide, since,among other reasons, concentrated solutions of hydrogen peroxide greaterthan 65% by weight can be hazardous due to the oxidizing nature thereof.

U.S. Pat. No. 4,952,370 to Cummings et al. discloses a sterilizationprocess wherein aqueous hydrogen peroxide vapor is first condensed onthe article to be sterilized, and then a source of vacuum is applied tothe sterilization chamber to evaporate the water and hydrogen peroxidefrom the article. This method is suitable to sterilize surfaces,however, it is ineffective at rapidly sterilizing diffusion-restrictedareas, such as those found in lumened devices, since it too depends onthe diffusion of the hydrogen peroxide vapor into the lumen to effectsterilization.

U.S. Pat. No. 4,943,414, entitled "Method for Vapor Sterilization ofArticles Having Lumens," and issued to Jacobs et al., discloses aprocess in which a vessel containing a small amount of a vaporizableliquid sterilant solution is attached to a lumen, and the sterilantvaporizes and flows directly into the lumen of the article as thepressure is reduced during the sterilization cycle. This system has theadvantage that the water and hydrogen peroxide vapor are pulled throughthe lumen by the pressure differential that exists, increasing thesterilization rate for lumens, but it has the disadvantage that thevessel needs to be attached to each lumen to be sterilized. In addition,water is vaporized faster and precedes the hydrogen peroxide vapor intothe lumen.

In U.S. Pat. No. 5,492,672, there is disclosed a process for sterilizingnarrow lumens. This process uses a multicomponent sterilant vapor andrequires successive alternating periods of flow of sterilant vapor anddiscontinuance of such flow. A complex apparatus is used to accomplishthe method. Because flow through of vapor is used, closed end lumens arenot readily sterilized in the process.

Thus, there remains a need for a simple and effective method of vaporsterilization of articles having areas where diffusion of these vaporsis restricted, such as long, narrow lumens.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method for sterilizingan interior of an article with a diffusion restricted area, such as anarticle having a lumen. The method includes the steps of contacting theinterior of the article with a source of peroxide, and exposing thearticle to negative pressure for a time period sufficient to effectcomplete sterilization. In one embodiment, the source of peroxidecomprises a liquid or condensed vapor. In another embodiment, the sourceof peroxide comprising a liquid comprises hydrogen peroxide or peraceticacid. In another embodiment, the source of peroxide comprising acondensed vapor comprises hydrogen peroxide or peracetic acid vapor. Ifthe exposing step is conducted for 1 hour at 40° C. and 10 torr, and thesource of peroxide comprises 1 mg/L hydrogen peroxide, the diffusionrestricted area preferably retains 0.17 mg/L or more hydrogen peroxide,or retains 17% or more of the hydrogen peroxide placed therein after theexposing step. In certain preferred embodiments, thediffusion-restricted area has the same or more diffusion restrictionthan provided by a lumen 27 cm in length and an internal diameter of 3mm, or has the same or more diffusion restriction than provided by alumen having a ratio of length to internal diameter greater than 50. Thesource of peroxide is preferably at a concentration of less than 25% byweight. The contacting step can be performed by delivery via a methodsuch as injection, static soak, liquid flow-through, aerosol spray,condensation or physical placement. In a preferred embodiment, thediffusion-restricted area is a lumen at least 27 cm in length and havingan internal diameter of no more than 3 mm, more preferably having aninternal diameter of no more than 1 mm. The exposing step is preferablyperformed for 60 minutes or less, and is preferably performed at apressure less than the vapor pressure of hydrogen peroxide. Thus, thepreferred pressure range under conditions of the present invention isbetween 0 and 100 torr. In one particularly preferred embodiment, thepressure is approximately 10 torr and the exposing step is conducted ata temperature of approximately 23° C. to approximately 28° C. Theexposing step can include the step of heating the article, such as byheating the chamber in which the exposing step occurs. The chamber canbe heated to about 40° C. to about 45° C. Alternatively, the source ofperoxide can be heated, such as to a temperature of about 40° C. toabout 45° C. Optionally, the step of exposing the device to a plasma canbe conducted during the step of exposing the device to negativepressure. In one embodiment employing exposure to plasma, the method isperformed within a first chamber and the plasma is generated in asecond, separate chamber. This embodiment further comprises the step offlowing the plasma into the first chamber. Advantageously, thecontacting and/or exposing steps of the method can be repeated one ormore times.

Another aspect of the present invention relates to a method forsterilizing an interior and an exterior of an article. This methodincludes the following steps: contacting the article with a source ofperoxide; and placing the article in a diffusion-restricted environment.The contacting and placing steps can be performed in either order. Thesesteps are followed by exposing the diffusion-restricted environment tonegative pressure for a time period sufficient to effect completesterilization. The contacting step can be performed both before andafter the placing step. If the exposing step is conducted at 40° C. and10 torr, and a source of peroxide comprising 1 mg/L of hydrogen peroxideis introduced, the diffusion restricted environment preferably retains0.17 mg/L or more hydrogen peroxide after the exposing step, or retains17% or more of the hydrogen peroxide placed therein after the exposingstep. The exposing step can include the step of heating the article,such as by heating the chamber in which the exposing step occurs or byheating the source of peroxide. In certain preferred embodiments, thediffusion-restricted environment has the same or more diffusionrestriction than provided by a single entry/exit port of 9 mm or less ininternal diameter and 1 cm or greater in length, or is sufficientlydiffusion restricted to completely sterilize a stainless steel bladewithin a 2.2 cm by 60 cm glass tube having a rubber stopper with a 1 mmby 50 cm stainless steel exit tube therein at a vacuum of 10 torr forone hour at 40° C. In one embodiment, the source of peroxide comprises aliquid or condensed vapor. In another embodiment, the source of peroxidecomprising a liquid comprises hydrogen peroxide or peracetic acid. Inanother embodiment, the source of peroxide comprising a condensed vaporcomprises hydrogen peroxide or peracetic acid vapor. The contacting stepcan be by delivery via a method such as injection, static soak, liquidflow-through, aerosol spray, condensation or physical placement. Plasmacan also be used during the step of exposing the lumen to negativepressure. If plasma is used, the method can be performed within a sealedchamber and the plasma generated within the container. Thus, the methodcan be performed within a first chamber and the plasma generated in asecond, separate chamber and the plasma flowed into the first chamber.The diffusion-restricted container can have at least one exit tube, suchas one that is at least 1.0 cm in length and has an internal diameter of9 mm or less. The exit tube can also include a filter. In a preferredembodiment, the filter is sufficient to prevent entry of bacteria fromthe environment into the container. The source of peroxide can be usedat a concentration of less than 25% by weight. The exposing step ispreferably performed for 60 minutes or less. The method can be conductedalong with the step of heating the article during the exposing step.Thus, the exposing step can be conducted within a chamber, and thechamber heated during the exposing step. The exposing step can beconducted at a negative pressure between 0 and 100 Torr. Advantageously,the various steps of this method can also be repeated one or more times.

Still one more aspect of the invention relates to a method for making asterilized article within a diffusion-restricted container. This methodincludes contacting the article with a source of peroxide, and placingthe article in the diffusion-restricted container in either order. Ifthe initial contacting step precedes the placing step, the contactingstep can be repeated after the placing step. These steps are followed byexposing the diffusion-restricted container to negative pressure for atime period sufficient to effect complete sterilization of the article.The container used in this aspect of the invention has at least one exittube. The exit tube preferably has a filter therein which is preferablysufficient to prevent entry of bacteria into the container. The exittube is at least 1.0 cm in length and/or has an internal diameter of 9mm or less. Advantageously, the exposing step, the contacting step, orthe entire method can be repeated one or more times. In a preferredembodiment, the contacting step comprises delivery via injection, staticsoak, liquid flow-through, aerosol spray, condensation or physicalplacement. The container can be exposed to a plasma during the step ofexposing the container to negative pressure. In one embodiment, themethod is performed within a sealed chamber and the plasma is generatedwithin the chamber. The exposing step is preferably performed for 60minutes or less and/or at a pressure between 0 and 100 Torr. Thecontainer can be heated during the exposing step, or the source ofperoxide heated prior to the contacting step. The invention alsoincludes the sterilized article within a diffusion-restricted containerproduced by the method of this aspect. In one embodiment, the source ofperoxide comprises a liquid or condensed vapor. In another embodiment,the source of peroxide comprising a liquid comprises hydrogen peroxideor peracetic acid. In another embodiment, the source of peroxidecomprising a condensed vapor comprises hydrogen peroxide or acetic acidvapor.

Still one more aspect of the invention relates to a method for making asterilized article within a diffusion-restricted container. This methodincludes placing the article in the diffusion-restricted container andcontacting the container with a source of peroxide, in either order.These steps are followed by exposing the diffusion-restricted containerto negative pressure for a time period sufficient to effect completesterilization of the article. The container used in this aspect of theinvention has at least one communication port comprising an exit tube orair and vapor permeable window. The exit tube preferably has a filtertherein which is preferably sufficient to prevent entry of bacteria intothe container. The exit tube is at least 1.0 cm in length and/or has aninternal diameter of 9 mm or less. The communication port is preferablyconnected through a connector to the article to be sterilized, so thatsterilant vapor may flow through the article and out of the container.The connector is preferably tubing or an adaptor which can be attachedto a lumen of said article, or an enclosure which contains a portion ofthe article with the lumen. In one embodiment, the exit tube isadditionally connected to a valve outside the container and the valve isconnected with a vacuum source. In one embodiment, the communicationport comprising a window is impermeable to microorganisms.Advantageously, the exposing step, the contacting step, or the entiremethod can be repeated one or more times. In a preferred embodiment, thecontacting step comprises delivery via injection, static soak, liquidflow-through, aerosol spray, condensation or physical placement. Thecontainer can be exposed to a plasma during the step of exposing thecontainer to negative pressure. In one embodiment, the method isperformed within a sealed chamber and the plasma is generated within thechamber. The exposing step is preferably performed for 60 minutes orless and/or at a pressure between 0 and 100 Torr. The container can beheated during the exposing step, or the source of peroxide heated priorto the contacting step. The invention also includes the sterilizedarticle within a diffusion-restricted container produced by the methodof this aspect. In one embodiment, the source of peroxide comprises aliquid, a solid or condensed vapor. In another embodiment, the source ofperoxide comprising a liquid comprises hydrogen peroxide or peraceticacid. In another embodiment, the source of peroxide comprising a solidcomprises a urea peroxide complex or sodium pyrophosphate peroxidecomplex or like complex. In another embodiment, the source of peroxidecomprising a condensed vapor comprises hydrogen peroxide or acetic acidvapor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional illustration of a lumen containing aninoculated stainless steel blade placed within a glass tube having onlya narrow opening to create a diffusion-restricted environment fortesting the sterilization method of the present invention.

FIG. 2 is a cross-sectional illustration of an inoculated stainlesssteel blade placed directly within a glass tube having only a narrowopening to create an alternate diffusion-restricted environment fortesting the sterilization method of the present invention.

FIG. 3 is a cross-sectional illustration of an inoculated stainlesssteel blade placed directly within a glass tube having a filter placedat its narrow opening to create an alternate diffusion-restrictedenvironment for testing the sterilization method of the presentinvention.

FIG. 4 is a cross-sectional illustration of one embodiment of adiffusion restricted environment represented by a container having alimited diffusion port (communication port consisting of tubing).

FIG. 5A is a cross-sectional illustration of one embodiment of adiffusion restricted environment represented by a container having alimited diffusion port (communication port consisting of tubing or thelumen device) and a tubing connector to connect a lumen device to thecommunication port of the container.

FIG. 5B is a cross-sectional illustration of one embodiment of adiffusion restricted environment represented by a container having alimited diffusion port (communication port consisting of tubing or thelumen device) and an enclosure connector to connect a lumen device tothe communication port of the container.

FIG. 6 is a cross-sectional illustration of one embodiment of adiffusion restricted environment represented by a container having alimited diffusion port and an enclosure connector to connect a lumendevice to the window.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Sterilizing the inside of lumened devices has always posed a challengeto sterilization systems. Achieving rapid sterilization of lumeneddevices or other diffusion restricted articles at low temperatures andlow concentrations of sterilant represents an even greater challenge. Inthe present invention, the shortcomings of the prior art sterilizationsystems are overcome by pre-treating or contacting articles to besterilized with a source of peroxide prior to exposure to a vacuum, oroptionally, plasma. Alternatively, a diffusion-restricted environmentcontaining articles to be sterilized can be contacted with a source ofperoxide prior to exposure to a vacuum. The source of peroxide comprisesa liquid or condensed vapor in the case wherein an article is contacted.In the case wherein a diffusion-restricted environment is contacted, thesource of peroxide additionally comprises a solid. The liquid comprisesaqueous solutions of hydrogen peroxide or peracetic acid. The solidcomprises a urea peroxide complex, or sodium pyrophosphate peroxidecomplex or like peroxide complex. The vapor comprises hydrogen peroxideor peracetic acid vapor. The preferred method of the present inventionutilizes aqueous hydrogen peroxide as the source of peroxide to contactan article to be sterilized. The methods of the present inventionprovide for the rapid sterilization of lumened and non-lumened articlesunder conditions that will not damage the articles nor leave toxicresidues on the sterile articles.

In the method of the present invention, the source of the peroxide isdelivered into direct contact with the article to be sterilized or withthe diffusion-restricted environment containing the article to besterilized. In the case of a lumened device, the source of peroxide maybe delivered directly into the lumen. In the case of an article havingan area where diffusion of vapor is restricted, the source of peroxidemay be delivered to the interior of the diffusion restricted area. Forarticles which are not diffusion-restricted, the source of peroxide canbe introduced anywhere into the diffusion-restricted environment. Thesource of peroxide is delivered into the lumen or into contact with thearticle to be sterilized or into contact with the diffusion-restrictedenvironment containing the article to be sterilized through means suchas direct delivery or physical placement, a static soaking process, aliquid flow-through process, by aerosol spray or by condensation of avapor. Physical placement also includes placement of a reservoircontaining the source of peroxide. In the preferred method of thepresent invention, the aqueous solutions of hydrogen peroxide can berelatively dilute, e.g., as low as 1-6% or lower by weight, sincesterilization is not achieved through contact with the hydrogen peroxidesolution, but rather, is achieved at low temperatures and in shortperiods of time upon exposure to hydrogen peroxide vapor under vacuum orvacuum combined with plasma. The method of the present invention isparticularly effective with articles having inaccessible orhard-to-reach places. Such articles include long, narrow lumens, hinges,and other articles having spaces where diffusion of vapors isrestricted.

The general operation of one embodiment of a preferred method of thepresent invention, which is useful for sterilizing the inside of long,narrow lumens, is as follows:

1. The lumen to be sterilized is contacted with a source of peroxide.The source of peroxide can be physically delivered as a small amountdirectly into the lumen, or by static soaking, liquid flow-through,aerosol spray or condensation of a vapor.

2. The lumen to be sterilized is placed within a chamber, and thechamber is sealed and evacuated. (The source of peroxide can also bedelivered to the inside of the article after placing the article in thechamber.)

3. The lumen is exposed to the vacuum for a period of time and at atemperature sufficient to effect sterilization.

4. The sterile lumen is removed from chamber.

In an alternative embodiment of the method of the present invention, asimilar method is used to sterilize both the inside and outside of anarticle. In this alternative embodiment, the article to be sterilized isplaced in a diffusion-restricted environment. The diffusion-restrictedenvironment can be a rigid container or flexible pouch having at leastone exit tube. In this embodiment, the exit tube is preferablydiffusion-restricted. Alternatively, it is not necessary that the exittube be diffusion-restricted so long as diffusion of sterilant vapor islimited by the article to be sterilized, such as the case whereinsterilant vapor must flow through a limited diffusion area or lumen ofan article to be sterilized. This depends upon the configuration of thecontainer. The exit tube may be constructed from a variety of materials,such as glass, metals and plastics and may include a filter. The filtermay be sufficient to prevent entry of bacteria from the environment intothe container. The source of peroxide is introduced to the inside of thearticle. The source of peroxide can be introduced either before or afterplacing the article in the diffusion-restricted environment. Thediffusion-restricted environment containing the article to be sterilizedis then placed in the chamber, exposed to vacuum and removed as in steps2 through 4 above.

The general operation of an alternative embodiment of the method of thepresent invention, which is also useful for sterilizing the inside oflong, narrow diffusion-restricted lumens, is as follows:

1. The article to be sterilized is placed in a diffusion-restrictedenvironment such as a container, said container comprising at least onecommunication port comprising an exit tube or air and vapor permeablewindow; and

2. The diffusion-restricted environment is contacted with a source ofperoxide, steps 1. and 2. being performed in either order; followed by

3. The diffusion-restricted environment is exposed to negative pressurefor a time period sufficient to effect complete sterilization of saidarticle.

The communication port is preferably connected through a connector tothe article, so that sterilant vapor may flow through the article andout of the container. In this embodiment, the communication portcomprising an exit tube or air and vapor permeable window is alsopreferably diffusion-restricted. Alternatively, it is not necessary thatthe communication port, in particular an air and vapor permeable window,be a limited diffusion port so long as diffusion of sterilant vapor islimited by the article to be sterilized, such as the case whereinsterilant vapor must flow through a limited diffusion area or lumen ofan article to be sterilized. This depends upon the configuration of thecontainer. The exit tube may be constructed from a variety of materials,such as glass, metals and plastics and may include a filter. The filtermay be sufficient to prevent entry of bacteria from the environment intothe container. The air and vapor permeable window may be constructedfrom permeable materials such as Tyvek.

In yet another alternative embodiment of the present invention whichpertains to all of the above methods, the article to be sterilized isexposed to a vacuum followed by low temperature plasma for a timesufficient to effect sterilization. When used in the presentspecification and claims, the term "plasma" is intended to include anyportion of the gas or vapor that contains electrons, ions, freeradicals, dissociated and/or excited atoms or molecules produced as aresult of an applied electric field, including any accompanyingradiation that might be produced. The applied field may cover a broadfrequency range; however, a radio frequency or microwaves are commonlyused.

The sterilization methods of the present invention can also be used withplasmas generated by the method disclosed in the previously mentionedU.S. Pat. No. 4,643,876. Alternatively, the methods of the presentinvention may be used with plasmas described in U.S. Pat. Nos. 5,115,166or 5,087,418, in which the article to be sterilized is located in achamber that is separated from the plasma source.

The present invention provides several advantages over earlier vaporsterilization systems, such as, (1) the rapid sterilization of lumeneddevices and diffusion restricted articles can be rapidly achieved at lowtemperatures; (2) the use of concentrated, potentially hazardous,solutions of anti-microbials is avoided; (3) the need to attach aspecial vessel to deliver sterilant vapors into long, narrow lumens iseliminated; (4) no toxic residues remain; (5) since the product is dryat the end of the process, sterile storage of these articles can beachieved; (6) closed end lumens can be sterilized; and (7) the processcan be repeated as desired without undue effects. The method of thepresent invention therefore provides for a highly efficient,nonhazardous, and relatively inexpensive method of sterilization.

To determine the efficacy of the preferred sterilization method of thepresent invention, preliminary tests were first performed to evaluatethe effect of dilute hydrogen peroxide solutions on contaminatedsurfaces in an open, non-diffusion restricted environment. These testsare described below in Example 1.

EXAMPLE 1

To evaluate the sterilization efficacy of dilute hydrogen peroxidesolution alone, a biological challenge consisting of 2.5×10⁶ Bacillusstearothermophilus spores on a stainless steel scalpel blade was used.Inoculated blades were submerged in 40 ml of hydrogen peroxide solutionin a 100 ml beaker. Four different concentrations of hydrogen peroxidesolution were used: 3%, 6%, 9% and 12% by weight. The blades wereallowed to soak in the peroxide solutions for various time periods. Theblades were then removed from the solution and tested for sterility. Theresults of this testing are listed in Table 1 as a ratio of the numberof inoculated blades which remain contaminated after treatment over thenumber of inoculated blades tested.

                  TABLE 1    ______________________________________    Effect of H.sub.2 O.sub.2 Concentration and Soak Times on    Sporicidal Activity of H.sub.2 O.sub.2 Solution              Concentration of H.sub.2 O.sub.2 Solution    Soak Time   3%      6%        9%    12%    ______________________________________     1 min      4/4     4/4       4/4   4/4     5 min      4/4     4/4       4/4   4/4    30 min      4/4     4/4       4/4   4/4    60 min      4/4     4/4       4/4   4/4    90 min       N/D*   4/4       2/4   0/4    120 min     N/D     4/4       N/D   N/D    ______________________________________     *N/D = not determined

Complete sterilization was not effected until after the blades had beensoaked in 12% hydrogen peroxide solution for at least 90 minutes.Moreover, none of the blades tested were sterilized after 2 hours in ⁶ %hydrogen peroxide solution. It is clear from these data that contactwith dilute hydrogen peroxide solution alone is ineffective at providingsterilization, unless extended soak times and concentrated solutions areused.

Testing was next performed to evaluate the effect on the sterilizationof long, narrow lumens of a pretreatment step in which the lumens to besterilized are exposed to hydrogen peroxide solution prior to exposureto a vacuum. The testing evaluated the efficacy of hydrogen peroxidevapor sterilization inside the lumens. The testing is detailed below inExample 2.

EXAMPLE 2

A biological challenge consisting of 1.9×10⁶ B. stearothermophilusspores on a stainless steel scalpel blade was used. Some inoculatedblades were pre-treated with a solution of aqueous hydrogen peroxide.Other inoculated blades, designated control blades, did not receivepretreatment with hydrogen peroxide. The pretreatment consisted of 5minutes of static soaking in peroxide solution. The pre-treated bladeswere blotted dry, and each blade was then placed inside a stainlesssteel lumen, 3 mm internal diameter (ID)×50 cm length. The lumen had acenter piece of 1.3 cm ID and 5 cm length. The pre-treated blade wasplaced inside this center piece, and additional hydrogen peroxidesolution was added into the center piece in various amounts. Controlblades were handled identically, except that they did not receivepretreatment with hydrogen peroxide solution. The lumens were placed ina vacuum chamber, and the chamber was evacuated to 1 Torr and held therefor 15 minutes, during which time the temperature increased fromapproximately 23° C. to approximately 28° C. Following exposure to thevacuum, the chamber was vented and the blades were removed from thechamber and tested for sterility. The results were as follows:

                  TABLE 2    ______________________________________    Effect of Pretreatment and Hydrogen Peroxide Concentration    on Sterilization of the Interior of Lumens    Additional peroxide added                  Blades not pre-treated                                Blades pre-treated in    into the center piece                  with peroxide peroxide solution    ______________________________________    (A) With 1% hydrogen peroxide solution and vacuum    10 μL      +             +    20 μL      +             +    30 μL      +             +    40 μL      +             +    50 μL      +             +    100 μL     +             -    150 μL     +             -    200 μL     -             -    250 μL     -             -    (B) With 3% hydrogen peroxide solution and vacuum    10 μL      -             -    20 μL      -             -    30 μL      -             -    40 μL      -             -    50 μL      -             -    100 μL     -             -    150 μL     -             -    200 μL     -             -    250 μL     -             -    (C) With 6% hydrogen peroxide solution and vacuum    10 μL      -             -    20 μL      -             -    30 μL      -             -    40 μL      -             -    50 μL      -             -    ______________________________________

As seen from these results, sterilization can be effected usingrelatively dilute solutions of peroxide and exposure to negativepressure. When the vacuum was applied, the peroxide added to the centerpiece of the lumen was vaporized and contacted the blade, which wassufficient to effect sterilization. It can be seen from these data thatthe pre-treatment increases effectiveness, but that pre-treatment isunnecessary as long as the peroxide diffuses from the inside to theoutside,.

Sterilization inside various lumen sizes after pretreatment withperoxide was compared with sterilization inside the lumens without thepretreatment step. This testing is detailed in Example 3.

EXAMPLE 3

A biological challenge consisting of 1.9×10⁶ B. stearothermophilusspores on a stainless steel scalpel blade was used. Test A in Table 3below consisted of the inoculated blades being pretreated with asolution of 3% aqueous hydrogen peroxide. The pretreatment consisted of5 minutes of static soaking in the peroxide solution. The pretreatedblades were blotted dry, then placed into the center piece of astainless steel lumen which varied in size, together with 10 μl of 3%hydrogen peroxide solution. The center piece was 1.3 cm ID and 5 cmlength. Test B in Table 3 below consisted of identically inoculatedcontrol blades which did not receive pretreatment with hydrogenperoxide. Each inoculated control blade was placed directly into thecenter piece of a stainless steel lumen together with 10 μl of 3%hydrogen peroxide solution. The center piece had dimensions identical tothose in Test A. Lumens of various dimensions were used to evaluate theeffect on sterilization of lumen internal diameter and length. Thelumens were placed in a vacuum chamber, and the chamber was evacuated to1 Torr for 15 minutes. During this 15 minutes of the sterilizationcycle, the temperature increased from approximately 23° C. toapproximately 28° C. Following exposure to the vacuum, the chamber wasvented and the blades were removed from the chamber and tested forsterility. The results are reported in Table 3, where "L/D Ratio"indicates the ratio of length to internal diameter.

                  TABLE 3    ______________________________________    Effect of Pretreatment With Dilute Hydrogen    Peroxide in Various Sized Lumens    SS lumen size L/D Ratio  Test A  Test B    ______________________________________    1 mm × 50 cm                  500        -       -    1 mm × 40 cm                  400        -       -    1 mm × 27 cm                  270        -       -    1 mm × 15 cm                  150        -       -    3 mm × 50 cm                  1662/3     -       -    3 mm × 40 cm                  133e,fra 1/3                             -       -    3 mm × 27 cm                   90        -       +    3 mm × 15 cm                   50        +       +    6 mm × 50 cm                   831/3     -       -    6 mm × 40 cm                   662/3     -       -    6 mm × 27 cm                   45        +       +    6 mm × 15 cm                   25        +       +    ______________________________________

All lumens having a L/D ratio greater than 50 which were tested underthe conditions of Test A of Example 3 were sufficientlydiffusion-restricted to be sterilized in this system. Thus, it isbelieved that other lumens having an L/D ratio greater than 50 shouldalso provide a sufficient level of diffusion-restriction forsterilization in accordance with the present invention. This testingshows that, in direct contrast to prior art methods, sterility throughdiffusion of hydrogen peroxide vapor from inside the article to outsidethe article is easier to achieve in longer, narrower lumens than inshorter, wider lumens. This is believed to be due to the larger lumensallowing too much of the hydrogen peroxide vapor to diffuse out of theinside of the lumen during the sterilization process. Thus, the vapordoes not contact the internal surfaces for a period of time sufficientor at a concentration sufficient to effect sterilization.

As discussed above, prior art methods of hydrogen peroxide vaporsterilization of lumens are generally limited to use on relatively shortand wide lumens. In contrast to these prior art methods, the method ofthe present invention is effective on the interior of long, narrowlumens, including those longer than 27 cm in length and/or having aninternal diameter of less than 3 mm.

To determine whether the ability of the sterilant vapor to diffusewithin the system is a critical factor in achieving sterility,additional testing was performed to compare diffusion restricted andopen, non-diffusion restricted systems. A non-diffusion restrictedsystem is one in which the diffusion of vapors in and around the articleis not restricted by narrow openings, long, narrow lumens, or the like.As used herein, "diffusion-restricted" refers to any one or more of thefollowing properties: (1) the ability of an article placed within thesterilization system of the present invention to retain 0.17 mg/L ormore hydrogen peroxide solution after one hour at 40° C. and 10 torr;(2) having the same or more diffusion restriction than provided by asingle entry/exit port of 9 mm or less in internal diameter and 1 cm orgreater in length; (3) having the same or more diffusion restrictionthan provided by a lumen 27 cm in length and having an internal diameterof 3 mm; (4) having the same or more diffusion restriction than providedby a lumen having a ratio of length to internal diameter greater than50; (5) the ability of an article placed within the sterilization systemof the present invention to retain 17% or more of the hydrogen peroxidesolution placed therein after one hour at 40° C. and 10 torr; or (6)being sufficiently diffusion-restricted to completely sterilize astainless steel blade within a 2.2 cm by 60 cm glass tube having arubber stopper with a 1 mm by 50 cm stainless steel exit tube therein ata vacuum of 10 torr for one hour at 40° C. in accordance with thepresent invention. It is acknowledged that characteristics (1) and (5)will vary depending on the initial concentration of hydrogen peroxideplaced into the article; however, this can be readily determined by onehaving ordinary skill in the art.

As discussed in the Background of the Invention, articles havingdiffusion restricted areas are difficult to sterilize using knownmethods of hydrogen peroxide vapor sterilization, since these methodsare dependent upon the diffusion of peroxide vapors from outside thearticle to the interior of the article. Testing performed to evaluatethe importance of sterilant vapor diffusion is described in Example 4.

EXAMPLE 4

Hydrogen peroxide vapor sterilization was tested in both open anddiffusion restricted systems. The open system consisted of stainlesssteel lumens having internal diameters of 1, 3, and 6 mm, and lengths of15, 27, 40 and 50 cm. Stainless steel scalpel blades were inoculatedwith 1.9×10⁶ B. stearothermophilus spores, and the blades placed in thecenter piece of the lumen together with 10 μl of 3% hydrogen peroxidesolution. The dimensions of the center piece were 1.3 cm ID, 5 cm lengthand 6.6 cc volume.

The diffusion restricted system is illustrated in FIG. 1. Identicallyinoculated scalpel blades 5 were placed within the center pieces 10 oflumens 15 having dimensions identical to those described above. Ten μlof 3% hydrogen peroxide solution was also added to the center piece 10of the lumen 15. The lumen 15 was then placed within a 2.2 cm×60 cmglass tube 20. The tube 20 was closed at one end, and the open end wasplugged with a rubber stopper 25 having a 1 mm×10 cm stainless steeltube 30 inserted through the stopper 25. Thus, gases entering or exitingthe glass tube 20 could pass only through this 1 mm 20×10 cm opening.

The open lumen system and the diffusion restricted system were placedinside a vacuum chamber. The chamber was evacuated to 1 Torr pressureand held there for 15 minutes, during which time the temperatureincreased from approximately 23° C. to approximately 28° C. The chamberwas then vented, and the blades removed from the lumens and tested forsterility. The results are as follows:

                  TABLE 4    ______________________________________    Hydrogen Peroxide Vapor Sterilization    in Open and Diffusion Restricted Systems            Peroxide    System  amount    Length  1 mm ID                                     3 mm ID                                            6 mm ID    ______________________________________    Open    10 μL of 3%                      50 cm   -      -      -                      40 cm   -      -      -                      27 cm   -      +      +                      15 cm   -      +      +    Diffusion            10 μL of 3%                      50 cm   -      -      -    Restricted        40 cm   -      -      -    Environment       27 cm   -      -      -                      15 cm   -      -      -    ______________________________________

Under the test conditions of Example 4, sterilization was not achievedin the shorter, wider lumens in the open system without pre-treatmentwith hydrogen peroxide. Pre-treatment, and other test conditions, suchas higher peroxide concentration or longer treatment time, would likelyallow sterilization of the 27 cm×3 mm lumen, which has an L/D ratiogreater than 50. In the diffusion restricted system, the blades weresterilized in all sizes of lumens, using a 3% hydrogen peroxidesolution.

These results indicate that providing a source of hydrogen peroxidewithin a diffusion restricted environment allows for completesterilization within the system. It is the restriction of vapordiffusion in the system, not the length or internal diameter of thelumen per se that determines the efficacy of the hydrogen peroxide vaporsterilization. Again, however, these data show that, unlike the priorart methods of hydrogen peroxide vapor sterilization of lumens, themethod of the present invention is effective even onnon-diffusion-restricted articles when placed into adiffusion-restricted environment.

To further test the idea that restriction of the diffusion of vapor in asystem affects the ability to sterilize the system, the followingexperiment was performed.

EXAMPLE 5

A stainless steel scalpel blade 5 was placed within a 2.2 cm×60 cm glasstube 20 which was closed at one end, as illustrated in FIG. 2. Eachblade 5 had been inoculated with 1.9×10⁶ B. stearothermophilus spores.For some of the testing, the glass tube 20 was left open at one end,providing an open system. To create a diffusion restricted environment,the open end of the glass tube 20 was sealed with a rubber stopper 25having a 1 mm×10 cm stainless steel tube 30 through its center. In boththe open and diffusion restricted systems, hydrogen peroxide solution ata concentration of either 3% or 6% was added to the glass tube 20 inamounts of 50, 100, 150 or 200 μl, together with the inoculated blade 5.The tube 20 was placed in a vacuum chamber, and the chamber evacuated to1 Torr for 15 minutes, during which time the temperature increased fromapproximately 23° C. to approximately 28° C. The diffusion restrictedsystem only was also tested at 1 Torr for 30 minutes, during which timethe temperature increased from approximately 23° C. to approximately 33°C. The vacuum chamber was then vented, and the blades 5 removed from thetube 20 and tested for sterility. The results are listed in Table 5below.

                  TABLE 5    ______________________________________    Hydrogen Peroxide Vapor Sterilization in    Open and Diffusion Restricted Systems             50 μL                  100 μL  150 μL                                     200 μL    ______________________________________    Open System, 15 minutes vacuum at 1 Torr:    3% peroxide               +      +          +     +    6% peroxide               +      +          +     +    Diffusion Restricted System, 15 minutes vacuum at 1 Torr:    3% peroxide               +      -          -     -    6% peroxide               -      -          -     -    Diffusion Restricted System, 30 minutes vacuum at 1 Torr:    3% peroxide               -      -          -     -    ______________________________________

These results show that the addition of hydrogen peroxide solution,followed by exposure to vacuum, is ineffective for achieving rapidsterilization in an open system. Identical treatment in a diffusionrestricted system, by comparison, results in complete sterilization,except at the very weakest concentration of hydrogen peroxide solutionin an amount of only 50 μl. Sterilization can be effected, however, byincreasing the exposure to the vacuum.

Thus, the method of the present invention, wherein small amounts ofhydrogen peroxide solution are delivered to the article to be sterilizedprior to exposure to a vacuum, is an effective method of sterilization.The method does not depend on the diffusion of sterilant vapor into thearticle being sterilized. Rather, the hydrogen peroxide vapor is createdby the vacuum within the system. This vapor is prevented from leavingthe system too quickly, because the diffusion of the sterilant vaporfrom the inside of the article to the outside of the article is slowed.In a diffusion restricted environment, the vapor therefore contacts thearticle to be sterilized for a period of time sufficient to effectcomplete sterilization. In addition, unlike the prior art methods wherethe water in the peroxide solution is vaporized first and becomes abarrier to the penetration of the peroxide vapor, the method of thepresent invention removes any water from the system first, therebyconcentrating the hydrogen peroxide vapor remaining in the system. Moreimportantly, in the preferred method of the present invention, thediffusion of vapor is from the inside to outside rather than outside toinside as in the prior art. As a result, diffusion-restriction in thepresent invention serves to increase the effectiveness of sterilizationrather than to decrease the effectiveness, as in the prior art.

To determine the effect of various pressures on a diffusion restrictedsterilization system, the following experiment was performed.

EXAMPLE 6

A stainless steel scalpel blade 5 was placed within a 2.2 cm×60 cm glasstube 20 which was closed at one end, as shown in FIG. 2. Each blade 5had been inoculated with 1.9×10⁶ B. stearothermophilus spores. To createa diffusion restricted environment, the open end of the glass tube 20was sealed with a rubber stopper 25 having a 1 mm×10 cm stainless steeltube 30 through its center. Hydrogen peroxide solution at aconcentration of 3% was added to the glass tube 20 in amounts of 50,100, 150 or 200 μl, together with the inoculated blade 5. The tube 20was placed in a vacuum chamber, and subjected to various pressures for15 minutes, during which time the temperature increased fromapproximately 23° C. to approximately 28° C. In a further experiment todetermine the effect of increased temperature on the system, the tube 20was first heated to 45° C., then subjected to 50 Torr pressure for 15minutes. The results were as follows.

                  TABLE 6    ______________________________________    Effect of Temperature and Pressure on a Diffusion Restricted System              50 μL                   100 μL  150 μL                                      200 μL    ______________________________________    15 minutes vacuum with 3% hydrogen peroxide solution:     1 torr pressure                +      -          -     -     5 torr pressure                -      -          -     -    10 torr pressure                -      -          -     -    15 torr pressure                -      -          -     -    20 torr pressure                -      -          -     -    25 torr pressure                -      -          -     -    30 torr pressure                +      +          +     +    35 torr pressure                +      +          +     +    40 torr pressure                +      +          +     +    45 torr pressure                +      +          +     +    50 torr pressure                +      +          +     +    15 minutes vacuum with 3% hydrogen peroxide at 45° C.:    50 torr pressure                -      -          -     -    ______________________________________

These data show that sterilization can be achieved in diffusionrestricted environments at pressures up to about 25 Torr at 28° C. Atpressures of 30 Torr and higher, sterilization was not achieved; this isbelieved to be due to the fact that the vapor pressure of hydrogenperoxide at 28° C. is approximately 28 Torr. Thus, at higher pressures,the liquid hydrogen peroxide inside the glass tube was not vaporizing.This was confirmed by the testing done at 50 Torr pressure at 45° C.,wherein sterilization was achieved. The vapor pressure of hydrogenperoxide is increased at 45° C., thus, the hydrogen peroxide wasvaporized at 50 Torr, effectively sterilizing the blade placed insidethe tube.

Accordingly, in order to achieve sterilization using the method of thepresent invention employing an aqueous solution of hydrogen peroxide,the temperature and pressure within the vacuum chamber should be suchthat vaporization of the aqueous hydrogen peroxide solution is achieved,i.e. the system should preferably be operated below the vapor pressureof the hydrogen peroxide. The pressure needs to be below the vaporpressure of hydrogen peroxide, such that the hydrogen peroxide solutionpresent in the system is vaporized and diffuses from the interior of thediffusion restricted environment to the outside. Alternatively, thehydrogen peroxide can be vaporized locally where the system remainsabove the vapor pressure by introducing energy to the site of theperoxide, such as through microwaves, radio waves, or other energysources.

To further determine the effect of varying the pressure and thetemperature in the diffusion restricted system described in Example 6,the following experiments were performed.

EXAMPLE 7

A stainless steel scalpel blade 5 was placed within a 2.2 cm×60 cm glasstube 20 which was closed at one end, as illustrated in FIG. 2. Eachblade 5 had been inoculated with 1.9×10⁶ B. stearothermophilus spores.To create a diffusion restricted environment, the open end of the glasstube 20 was sealed with a rubber stopper 25 having a 1 mm×10 cmstainless steel tube 30 through its center. Hydrogen peroxide solutionat a concentration of 3% was added to the glass tube 20 in amounts of50, 100, 150 or 200 μl together with the inoculated blade 5. The tube 20was placed in a vacuum chamber, and the chamber evacuated to 5 Torr. Tovary the pressure within the chamber, the valve to the vacuum pump wasclosed, such that the pressure within the chamber rose from 5 Torr to6.15 Torr after 15 minutes, during which time the temperature increasedfrom approximately 23° C. to approximately 28° C. In a second test, thetube 20 was placed in the chamber and the chamber was evacuated to 50Torr. The temperature of the glass tube 20 was increased to 45° C. afterthe evacuation of the chamber was complete. The tube 20 was treated for15 minutes. The results of these tests are reported below.

                  TABLE 7    ______________________________________    Effect of Varying Temperature and Pressure on    Diffusion Restricted Sterilization System               50 μL                    100 μL  150 μL                                       200 μL    ______________________________________    Pressure increased from 5 Torr to 6.15 Torr:    Efficacy Results                 -      -          -     -    Temperature of the tube increased to 45° C.:    Efficacy Results                 -      -          -     -    ______________________________________

These results show that maintaining a constant pressure or temperatureis not required in the diffusion restricted environment to effectsterilization. Under the conditions tested, the hydrogen peroxide isvaporized and kept in contact with the device to be sterilized for atime sufficient to effect complete sterilization.

The preferred method of the present invention relies on the delivery ofliquid hydrogen peroxide to the article to be sterilized prior to vacuumor plasma treatment. The following testing was performed to determinethe effect of the location of the delivery of the hydrogen peroxidewithin the diffusion restricted environment.

EXAMPLE 8

A stainless steel scalpel blade 5 was inoculated with 1.9×10⁶ B.stearothermophilus spores, and the blade 5 placed in the center piece 10of a lumen 15 as illustrated in FIG. 1. The dimensions of the centerpiece 10 were 1.3 cm ID, 5 cm length and 6.6 cc volume, while the lumenitself varied in size, having an ID of 1, 3 or 6 mm, and a length of 15,27, 40 or 50 cm. The lumen 15 was placed within a 2.2 cm×60 cm glasstube 20. The tube 20 was closed at one end, and the open end was pluggedwith a rubber stopper 25 having a 1 mm×10 cm stainless steel tube 30placed through the stopper 25. Thus, gases entering or exiting the glasstube 20 could pass only through this 1 mm×10 cm opening. 10 μl of 3%hydrogen peroxide solution was placed inside the lumen 15, or 100 μl of3% hydrogen peroxide solution was placed inside the glass tube 20, butoutside the stainless steel lumen 15. The glass tube 20 was then placedin a vacuum chamber, which was sealed and evacuated to 1 Torr for 15minutes, during which time the temperature increased from approximately23° C. to approximately 28° C. Results of this testing are as follows.

                  TABLE 8    ______________________________________    Effect of Hydrogen Peroxide Solution    Placed Outside Inner Lumen    Peroxide amount               Length  1 mm ID   3 mm ID 6 mm ID    ______________________________________    10 μL of 3%               50 cm   -         -       -    in lumen   40 cm   -         -       -               27 cm   -         -       -               15 cm   -         -       -    100 μL of 3%               50 cm   +         +       +    in glass tube               40 cm   +         +       +               27 cm   +         +       +               15 cm   +         +       -    ______________________________________

These data show that, under the test conditions of Example 8,sterilization did not occur within the inner lumen when the hydrogenperoxide solution was placed outside the lumen in a diffusion restrictedenvironment, but that complete sterilization was effected when thehydrogen peroxide solution was placed inside all of the lumens in adiffusion restricted environment. When the hydrogen peroxide vapor mustdiffuse from outside to inside, the sterilant vapor cannot enter theinner lumen in a diffusion restricted environment unless the lumen issufficiently large. Thus, when the hydrogen peroxide solution was placedoutside the lumen, only the shortest, widest lumens allowed sufficientvapor penetration to allow sterilization inside the lumen. These dataconfirm that prior art methods which require diffusion of sterilantvapor from outside the article to the interior article cannot achievesterilization in diffusion restricted environments under theseconditions. In contrast, under the same conditions except where thehydrogen peroxide was placed inside the article, allowing hydrogenperoxide to diffuse from inside to outside, complete sterilizationoccurred with much lower amounts of hydrogen peroxide.

The method of the present invention is therefore useful in environmentswhere diffusion of the sterilant vapor is limited. To evaluate theeffect of changes in the amount of diffusion restriction within adiffusion restricted environment, the following testing was performed.

EXAMPLE 9

A stainless steel scalpel blade 5 was inoculated with 1.9×10⁶ B.stearothermophilus spores, and placed in a 2.2 cm×60 cm glass tube 20 asillustrated in FIG. 2. The tube 20 was closed at one end, and the openend was plugged with a rubber stopper 25. Stainless steel tubing 30 ofvarious dimensions was inserted through the stopper 25. Thus, gasesentering or exiting the glass tube 20 could pass only through theopening in the tubing 30, which varied from 1 mm to 6 mm in diameter.Three percent hydrogen peroxide solution in volumes ranging from 50 μLto 200 μL was also placed inside the glass tube 20. The glass tube 20was then placed in a vacuum chamber, which was sealed and evacuated to 5Torr for 15 minutes, during which time the temperature increased fromapproximately 23° C. to approximately 28° C. In addition, three lumenswere tested at 10 Torr for 15 minutes with 3% hydrogen peroxide. Theresults of this testing are listed below in Table 9.

                  TABLE 9    ______________________________________    Effects of Tubing Dimension and    Vacuum Pressure on Sterilization    ______________________________________    15 minutes vacuum at 5 Torr with 3% hydrogen peroxide    SS tubing   50 μL                       100 μL  150 μL                                        200 μL    ______________________________________    1 mm × 10 cm                -      -          -     -    1 mm × 5 cm                -      -          -     -    1 mm × 2.5 cm                +      -          -     -    3 mm × 10 cm                -      -          -     -    3 mm × 5 cm                -      -          -     -    3 mm × 2.5 cm                +      -          -     -    6 mm × 10 cm                -      -          -     -    6 mm × 5 cm                +      -          -     -    6 mm × 2.5 cm                +      -          -     -    ______________________________________    15 minutes vacuum at 10 Torr with 3% hydrogen peroxide           SS tubing                    50 μL    ______________________________________           1 mm × 2.5 cm                    -           3 mm × 2.5 cm                    -           6 mm × 2.5 cm                    -    ______________________________________

Complete sterilization was achieved in the majority of the environmentstested. Sterilization could not be achieved at 5 torr using the shortestlength of stainless steel tubing and only 50 μl hydrogen peroxidesolution. Greater volumes of hydrogen peroxide must be used in thesesystems.

These data also confirm that the vacuum pressure affects sterilizationefficacy, since the container with the shortest and widest exit tubecould provide sterilization at 10 Torr, but not at 5 Torr. At too lowpressures (such as pressures below 5 Torr in the conditions tested)however, it appears that the hydrogen peroxide vapor is pulled from theinterior of the article being sterilized too quickly, resulting in aninsufficient amount of hydrogen peroxide vapor being allowed to contactthe interior of the device to effect sterilization. It would appear thatalthough a pressure of 5 torr produces acceptable results, a pressure ofapproximately 10 Torr is better under the conditions tested.

The method of the present invention has been shown to be effective indiffusion restricted environments of metal and glass. To evaluatewhether the method is effective in diffusion restricted environmentsformed of other materials, the experiments described in Examples 10 and11 were performed.

EXAMPLE 10

For this testing, a diffusion restricted system was tested. 1.2×10⁶ B.stearothermophilus spores were inoculated onto non-woven polypropylenepieces. As illustrated in FIG. 1, the inoculated pieces 5 were placedinside the center piece 10 of a plastic lumen 15, together with 10 μl of3% hydrogen peroxide solution. The center piece 10 was made of Teflon™and had dimensions of 1.3 cm ×5 cm. The lumen 15 varied from 1 mm to 6mm ID, and 15 cm to 50 cm in length. Teflon™ was used for the 1 mmlumen, polyethylene was used for the 3 mm and 6 mm lumen. The lumen 15was then placed within a 2.2 cm×60 cm glass tube 20. The glass tube 20was closed on one end, and the open end was sealed with a rubber stopper25 having a 1 mm×10 cm piece of PTFE tubing 30 through it. The glasstube 20 was placed in the vacuum chamber and treated for 15 minutes at 1Torr, during which time the temperature increased from approximately 23°C. to approximately 28° C. The results of this testing are set forthbelow.

                  TABLE 10A    ______________________________________    Sterilization in Diffusion Restricted Systems    Using Plastic Lumens    System    Pressure                      Length  1 mm ID                                     3 mm ID                                            6 mm ID    ______________________________________    Diffusion 1 torr  50 cm   -      -      -    Restricted System 40 cm   -      -      -                      27 cm   -      -      -                      15 cm   -      -      -    ______________________________________

Sterilization in diffusion restricted environments can be effected inboth short, wide lumens and long, narrow lumens, regardless of whethermetal or plastic is used to form the lumens. Thus, the method of thepresent invention is an effective sterilization method for diffusionrestricted articles, and can be used on a wide variety of such articles,regardless of their composition.

To further confirm this, 2.1×10⁶ B. stearothermophilus spores wereinoculated on stainless steel blades, and 1.2×10⁶ B. stearothermophilusspores were inoculated onto non-woven polypropylene pieces. As shown inFIG. 2, the blades 5 or non-woven polypropylene pieces 5 were placedinside a 2.2 cm×60 cm glass tube 20 together with 50 μl of 3% hydrogenperoxide solution. One end of the tube was closed, and the open end wassealed with a rubber stopper 25 having either a 1 mm×10 cm stainlesssteel tube 30 therein, or a 1 mm×10 cm piece of Teflon™ tubing 30therein. The glass tube 20 was placed inside a vacuum chamber andtreated for 15 minutes at 5 Torr, during which time the temperatureincreased from approximately 23° C. to approximately 28° C. The resultsare as follows.

                  TABLE 10B    ______________________________________    Effect of Metal and Plastic on Sterilization    in a Diffusion Restricted System                   SS tubing                            Teflon tubing    ______________________________________    Metal blade    -        -    Polypropylene  -        -    ______________________________________

Thus, all four combinations of metal and plastic provide for effectivehydrogen peroxide vapor sterilization in a diffusion restrictedenvironment. This testing confirms that the method of the presentinvention is an effective sterilization method for diffusion restrictedarticles, and can be used on a wide variety of such articles, regardlessof the materials used to form them.

Further testing was next performed to evaluate the effect of varioustemperatures and pressures on the sterilization of a diffusionrestricted system. The testing is described below.

EXAMPLE 11

Stainless steel blades were inoculated with 2.1×10⁶ B.stearothermophilus spores. The blades 5 were placed inside a 2.2 cm×60cm glass tube 20 as illustrated in FIG. 2, along with various amounts of3% hydrogen peroxide solution. The glass tube 20 was placed in a vacuumchamber and subjected to different pressures and different temperaturesfor various periods of time. During the sterilization cycles reported inTable 11A, the temperature increased from approximately 23° C. to thetemperatures indicated. In the experiments reported in Table 11B, thechamber was heated to approximately 45° C. In an alternative embodiment,rather than heating the chamber, the temperature of the peroxidesolution itself can be heated. In the experiments reported in Table 11C,the temperature increased from approximately 23° C. to approximately 28°C. during the 15 minute period of exposure to vacuum.

                  TABLE 11A    ______________________________________    Effect of Time and Volume of Peroxide on    Sterilization in a Diffusion Restricted Environment              5 min.              (approx.                    10 min.      15 min.              24° C.)                    (approx. 26° C.)                                 (approx. 28° C.)    ______________________________________    At 5 Torr pressure:     50 μL of 3% peroxide                -       -            -    100 μL of 3% peroxide                -       -            -    150 μL of 3% peroxide                +       -            -    200 μL of 3% peroxide                +       -            -    ______________________________________

                  TABLE 11B    ______________________________________    Effect of Elevated Chamber Temperature and Volume of Peroxide on    Sterilization in a Diffusion Restricted Environment                    5 min.    ______________________________________    Chamber at approximately 45° C.:    150 μL of 3% peroxide                      -    200 μL of 3% peroxide                      -    ______________________________________

                  TABLE 11C    ______________________________________    Effect of Pressure and Volume of Peroxide on    Sterilization in a Diffusion Restricted Environment    Approx. 28° C.                  1 torr      5 torr 10 torr    ______________________________________    With 15 minutes exposure time:     20 μL of 3% peroxide                  N/D         +      -     50 μL of 3% peroxide                  +           -      -    100 μL of 3% peroxide                  -           -      -    ______________________________________

Under the test conditions of Example 11, large volumes of hydrogenperoxide solution were ineffective at achieving sterilization whenvacuum was applied for only very short periods of time. This is believedto be at least partially because water vaporizes more quickly thanhydrogen peroxide. Thus, the water present in the aqueous solution willvaporize first, and more time is needed to vaporize the hydrogenperoxide. This also explains why the larger volumes of hydrogen peroxidesolution were effective at achieving sterilization at highertemperatures; the vaporization of the hydrogen peroxide occurs sooner athigher temperatures. Thus, when more water is present in the system,either higher temperatures or more time is required to achievesterilization.

Again, it would appear from these data that slightly higher pressures,i.e. 10 Torr, achieve more effective sterilization under theseconditions. This is believed to be because at higher pressures, morehydrogen peroxide vapor is retained inside the system. At too low apressure, the hydrogen peroxide vapor is pulled out of the system tooquickly.

In order to evaluate a putative minimum concentration of peroxide in theliquid/vacuum system in a diffusion-restricted container, Example 12 wascarried out.

EXAMPLE 12

Various concentrations of peroxide were used in a system substantiallyas described in connection with FIG. 2. In this system, the exit tube 35was a stainless steel tube having a length of 50 cm and an internaldiameter of 1 mm. A stainless steel blade inoculated with 1.9×10⁶ sporesof B. stearothermophilus was placed within the container which was a 2.2cm×60 cm glass tube. Various amounts of 3% hydrogen peroxide wereintroduced into the container. The container was placed in a vacuumchamber of 173 liters, and the pressure reduced to 10 Torr for a periodof one hour, during which time the temperature increased fromapproximately23° C. to approximately 40° C. Sporicidal activity wasevaluated at each concentration of peroxide. In addition, the amount ofperoxide remaining in the container after the sterilization process wasevaluated by standard titration techniques, whereby the peroxide wasreacted with potassium iodide and titrated with sodium thiosulfate.Results are shown in Table 12 where "N/D" indicates not determined.

                  TABLE 12    ______________________________________    Amount of Peroxide                     Sporicidal                              Remaining    in Glass Tube    Activity Peroxide    ______________________________________    0.5 mg/L liquid  +        N/D    0.6 mg/L liquid  +        N/D    0.7 mg/L liquid  +        N/D    0.8 mg/L liquid  +        N/D    0.9 mg/L liquid  +        N/D    1.0 mg/L liquid  -        0.17 mg/L    ______________________________________

The results reported in Table 12 indicate that 1.0 mg/L of 3% liquidperoxide were required in the system tested to effect sterilization.Further, under the conditions tested, a concentration of 0.17 mg/L ofperoxide remaining in the system was sufficient to provide completesterilization. These data also show that the glass tube used in theseexperiments provided a sufficient level of diffusion restriction toretain 17% of the hydrogen peroxide placed therein.

We further investigated the effects of length and internal diameter ofthe exit tube used in a system similar to that of Example 12. Thistesting is shown in Example 13.

EXAMPLE 13

A system similar to that described above in connection with Example 12,with the exception that 15 minutes of vacuum rather than one hour wasused.

Thus, the temperature increased only to about 28° C. In this testing,the size of the exit tube 35 was varied, as well as the volume of 3%peroxide solution. The results are reported below in Table 13.

                  TABLE 13    ______________________________________                50 μL                      100 μL                               150 μL                                        200 μL    ______________________________________    Open without tubing                  +       +        +      +     6 mm ID × 1 cm Length                  +       -        -      -     9 mm ID × 1 cm Length                  +       -        -      -    13 mm ID × 1 cm Length                  +       +        +      +    ______________________________________

The results show that provided sufficient peroxide is present, thediffusion-restriction provided by a single entry/exit port of 9 mm orless in internal diameter, or 1 cm or greater in length is sufficient toeffect sterilization.

To further evaluate the effect on sterilization efficacy of changes inthe amount of restriction of vapor diffusion in the system, thefollowing testing was performed.

EXAMPLE 14

A stainless steel blade was inoculated with 2.1×10⁶ B.stearothermophilus spores. The blade 5 was placed inside a 2.2 cm×60 cmglass tube 20 as shown in FIG. 3, together with various amounts of 3%hydrogen peroxide solution. One end of the tube was closed, and the openend was sealed with a rubber stopper 25 having a syringe filter 35inserted therein. The glass tube 20 was placed inside a vacuum chamberand treated for 15 minutes at 5 Torr, during which time the temperatureincreased from approximately 23° C. to approximately 28° C. As acontrol, identically inoculated blades were placed inside 2.2 cm×60 cmglass tubes. The open end of the tubes was left open, no stopper orsyringe filter was used. Thus, the diffusion of vapor from the interiorof the tube was not restricted.

Various syringe filters having various pore sizes were tested, includingMFS PTFE 25 mm syringe filters with a 0.2 μm membrane filter and a 0.5μm membrane filter; a Nalgene PTFE 50 mm syringe filter with a 0.2 μmmembrane filter and a 0.45 μm membrane filter; a Whatman Anotop™ 10 Plussterile syringe filter with a 0.02 μm membrane filter and a 0.1 μmmembrane filter; and finally, a Gelman Acrodisc™ CR PTFE syringe filterwith a 0.2 μm, 0.45 μm, and a 1.0 μm membrane. The results are asfollows.

                  TABLE 14    ______________________________________    Sporicidal Activity of H.sub.2 O.sub.2 Solution with Vacuum    in a Container Having a Syringe Filter            50 μL                 100 μL   150 μL                                     200 μL    ______________________________________    15 minutes vacuum and 3% hydrogen peroxide:    (a) Without syringe filter and stopper:     5 Torr   +      +           +     +    10 Torr   +      +           +     +    (b) With MFS ™ PTFE 25 mm syringe filter:    (1) 0.2 μm membrane filter     5 Torr   +      -           -     -    10 Torr   -      -           -     -    (2) 0.5 μm membrane filter     5 Torr   +      -           -     -    10 Torr   -      -           -     -    ______________________________________                   50 μL    ______________________________________    (3) With 2 MFS ™ filters together at 5 Torr pressure    Two 0.2 μm filters                     -    Two 0.5 μm filters                     -    ______________________________________            50 μL                 100 μL   150 μL                                     200 μL    ______________________________________    (c) With Nalgene ™ PTFE 50 mm syringe filter:    (1) 0.2 pm membrane filter     5 Torr   -      -           -     -    10 Torr   -      -           -     -    (2) 0.45 μm membrane filter     5 Torr   -      -           -     -    10 Torr   -      -           -     -    ______________________________________                  50 μL                       100 μL    ______________________________________    (d) With Whatman Anotop ™ 10 Plus syringe filter:    (1) 0.02 μm membrane filter     5 Torr         -      -    10 Torr         -      -    (2) 0.1 μm membrane filter     5 Torr         -      -    10 Torr         -      -    (e) With Gelman Acrodisc ™ CR PTFE syringe filter:    (1) 0.2 μm membrane filter     5 Torr         +      -    10 Torr         -      -    (2) 0.45 μm membrane filter     5 Torr         +      -    10 Torr         -      -    (3) 1.0 μm membrane filter     5 Torr         +      -    10 Torr         -      -    ______________________________________

As is apparent from these results, certain brands of filters do notcreate a sufficiently diffusion restricted environment at 5 Torrpressure when only 50 μL of hydrogen peroxide solution is placed in thesystem. Other brands of filters did provide sufficient diffusionrestriction; these brands of filters had either longer lumens or smallerfilter pore size. Using larger volumes of peroxide solution, 10 Torrpressure, or serial filters enhances the efficacy of the sterilizationsystem. This is important, as filters, including ones made of Tyvek™,are often used in packaging of sterile articles to preventrecontamination with bacteria. These filters generally have a pore sizeof 1 μm or less, or in the case of Tyvek™, create a tortuous path whichbacteria cannot cross. In the present invention, filters can be used incombination with other packaging means to create a diffusion restrictedenvironment to effect sterilization, and the sterile article can remaininside the packaging during storage prior to use; the filter willprevent re-contamination of the sterile article.

FIG. 4 is a cross-sectional illustration of one embodiment of adiffusion restricted environment represented by a container having alimited diffusion port or communication port consisting of tubing. Thiscommunication port 30 may have an air permeable microorganism barriersuch as a filter in order to maintain a sterility of the devices 15 and40 in the container 20 after the container 20 is removed from the vacuumsource. The non-lumen device 40 and the exterior of the lumen device 15can be sterilized with the peroxide vapor generated from the source ofperoxide within the container 20. In one method of efficientlysterilizing the interior of the lumen device 15, the peroxide vaporneeds to be generated within the lumen device 15. Therefore, the lumendevice 15 needs to be pre-treated with liquid peroxide.

FIGS. 5-6 illustrate other embodiments of the present inventionemploying other packaging means to create a diffusion-restrictedenvironment to effect sterilization. Another approach can be used tosterilize the interior of lumen device 15 without pre-treating theinterior of lumen device 15 with the source of peroxide. In order toflow the peroxide vapor generated inside container 20 through theinterior of lumen device 15, a connector can be used to connect thelumen device 15 to the communication port 30 of the container 20. FIGS.5A and 5B illustrate this approach. FIG. 5A is a cross-sectionalillustration of one embodiment of a diffusion restricted environmentrepresented by a container 20 having a limited diffusion port orcommunication port 30, consisting of tubing, and a tubing connector 45to connect a lumen device 15 to the communication port 30 of thecontainer 20. FIG. 5B is a cross-sectional illustration of oneembodiment of a diffusion restricted environment represented by acontainer 20 having a limited diffusion port (communication port 30consisting of tubing) and an enclosure connector 50 to connect a lumendevice 15 to the communication port 30 of the container 20. Theenclosure connector 50 has an interface 51 between the container 20 andthe enclosure connector 50. This interface 51 can be constructed inseveral different ways so as to allow a portion of the lumen device 15to be inserted into the connector enclosure 50, while maintaining an airand vapor pressure seal between parts 15 and 50. One way to achieve thisis with a camera shutter approach employing an iris diaphragm, such as aprecision iris diaphragm from Edmund Scientific. An optional spring canbe used to insure the closure of the shutter. Another way to achieve anacceptable interface is to employ two plates, wherein the area betweenthe two plates has a compressible material, such as a rubber material.The lumen device 15 can be placed between the two plates and the twoplates moved together to form a gas and vapor impermeable seal aroundthe lumen device 15. Optionally, a porous material like a sponge or airpermeable material may be utilized for the compressible material. Inthis case, some vapor sterilant can diffuse between the compressiblematerial and the lumen device. However, most of the sterilant vapor isforced to diffuse through the lumen device. Yet another way to achievean acceptable interface is to employ a hole or horizontal opening forone or more lumen devices 15, said hole or opening being a gas or liquidinflatable port. Thus, the connector can be a tubing adapter 45 whichcan be attached to the lumen device 15 or an enclosure 50 which containsa portion of the lumen device 15. Since one of the openings of the lumendevice 15 is connected to the communication port 30 with the connector45 or 50, the vaporized peroxide has to be evacuated through the lumendevice 15. Tubing connector 45 can be constructed of any materials suchas silicone, teflon, etc. which meet the thermal, pressure, gas andvapor compatibility requirements of the system. These sameconsiderations apply to materials utilized for other parts illustratedherein. Note that the limited diffusion port can be created by eitherthe communication port 30 or the lumen device 15.

FIG. 6 illustrates another possible arrangement. FIG. 6 is across-sectional illustration of one embodiment of a diffusion restrictedenvironment represented by a container 20 having a communication port 30consisting of a window with an air permeable barrier and an enclosureconnector 50 to connect a lumen device 15 to the window 30. In thisembodiment, the lumen device 15 is connected to the connector 50 and isused as the device to create the diffusion restricted area in thecontainer 20. Therefore, the communication port 30 in FIGS. 4, 5A and 5Bcan be replaced with an air permeable window 30 if desired. This porouswindow 30 allows the diffusion of air and vapor, but preventsmicroorganisms from outside from contaminating the sterilizedinstruments 15 or 40 in the container or pouch 20. Under the reducedpressure environment, the peroxide vapor is first generated in thecontainer or pouch 20 and then diffuses through the lumen device 15 intothe connector 50. The entire connector 50 can be made of air permeablematerial. FIG. 6 additionally illustrates how the reduced pressure is tobe achieved. This is achieved via a port 55 in the vacuum chamber 65,said port being connected to a vacuum pump 60 to produce the reducedpressure environment. In order to test whether other sterilants can alsobe used to effect sterilization in diffusion restricted environments,the following testing was performed.

EXAMPLE 15

A stainless steel blade was inoculated with 1.9×10⁶ B.stearothermophilus spores. The blade 5 was placed inside a 2.2 cm×60 cmglass tube 20 as shown in FIG. 2, along with various amounts of 4.74%peracetic acid solution (Solvay Interox Ltd., Warrington, England). Theglass tube 20 was placed in a vacuum chamber and subjected to 5 Torrpressure for 15 minutes, during which time the temperature increasedfrom approximately 23° C. to approximately 28° C. The results of thistesting is shown below.

                  TABLE 15    ______________________________________    Sterilization With Peracetic Acid in    a Diffusion Restricted System               50 μL                    100 μL 150 μL                                      200 μL    ______________________________________    Efficacy Results                 -      -         -     -    ______________________________________

These results show that peracetic acid, in which hydrogen peroxidecoexists, can also be used in the sterilization method of the presentinvention.

It was discovered that by delivering small amounts of hydrogen peroxidesolution to an article to be sterilized prior to exposure to vacuum,sterilization could be effected at lower temperatures and in shortperiods of time. The following testing was performed to evaluatedifferent methods of delivering hydrogen peroxide solution to thearticle to be sterilized. Further, the efficacy of vacuum treatment andplasma treatment following pretreatment with aqueous hydrogen peroxidewere compared. The testing is described in Example 16 below.

EXAMPLE 16

In a first series of tests, stainless steel blades were inoculated with2.5×10⁶ B. stearothermophilus spores. The blades were placed in theexpanded center piece of a 3 mm×50 cm stainless steel lumen. The lumenwas placed in a 1000 ml beaker containing 800 ml of hydrogen peroxidesolution. The lumen was soaked for 5 minutes in 3% hydrogen peroxidesolution. The number of surviving organisms following this initial soakwas determined. The lumens were removed from the hydrogen peroxidesolution and the outside blotted dry with paper towels. The inside ofthe lumens were dried by placing one end of the lumen into a flask andblowing with a three second burst of compressed air. The lumens wereshaken, and the blowing and shaking repeated until no more solution wasblown out. Subsequently, the lumen was placed in a sterilization chamberand exposed to either a vacuum of 0.5 Torr for 15 minutes, or plasma for15 minutes at 0.5 Torr. After 15 minutes of vacuum, the temperatureincreased from approximately 23° C. to approximately 28° C. The resultsare set forth below in Table 16A.

                  TABLE 16A    ______________________________________    Effect of H.sub.2 O.sub.2 Solution Soak on    Sporicidal Activity in Stainless Steel Lumens Prior to    Either a Plasma or a Vacuum Treatment             Number of             Surviving Sterility Test Results    Conc. H.sub.2 O.sub.2 (%)               Organisms After                           Soak     Soak +                                          Soak +    Soak Time 5 min               Soaking Alone                           Alone    Vacuum                                          Plasma    ______________________________________    3.0        8.2 × 10.sup.5                           4/4      0/4   0/4    ______________________________________

A five minute soak in 3% hydrogen peroxide solution was an effectivemeans for delivering the hydrogen peroxide into the lumen prior tovacuum or plasma treatment. As noted before, treatment with hydrogenperoxide solution only is ineffective to achieve sterilization usingdilute solutions and short soak times. Delivery of hydrogen peroxidesolution via static soaking is at least as effective a way to deliverthe hydrogen peroxide as depositing small volumes directly into thelumen of the device.

Flow-through delivery of hydrogen peroxide was tested next. Here,stainless steel blades were inoculated with 2.5×10⁶ B.stearothermophilus spores. The blades were placed in the expanded centerpiece of a 3 mm×50 cm stainless steel lumen. Hydrogen peroxide solutionat 3% concentration was delivered to the lumen at a flow rate of 0.1L/min, using a peristaltic pump. The lumen was dried as described above.Following pretreatment with hydrogen peroxide solution, the lumen wasthen placed in a sterilization chamber and exposed to either a vacuum of0.5 Torr for 15 minutes, or plasma for 15 minutes at 0.5 Torr. Theresults are set forth below in Table 16B.

                  TABLE 16B    ______________________________________    Effects of Flow-Through Delivery of H.sub.2 O.sub.2 Solution on    Sporicidal Activity Prior to Either a Vacuum or a Plasma    Treatment in Stainless Steel Lumens    Conc. H.sub.2 O.sub.2              Number of Surviving                              Sterility Test Results    (%)       Organisms after Flow +   Flow +    5 min flow              Flow Alone      Vacuum   Plasma    ______________________________________    3         6.2 × 10.sup.5                              0/4      0/4    ______________________________________

Delivery of the hydrogen peroxide solution via constant flow is also aneffective way to deliver hydrogen peroxide to the system.

Finally, the effect of delivery of hydrogen peroxide by aerosol spraywas tested. Stainless steel blades were inoculated with 2.5×10⁶ B.stearothermophilus spores. The inoculated blades were placed in theexpanded center piece of a 3 mm ×50 cm stainless steel lumen. Threepercent hydrogen peroxide solution was delivered to the lumen via a 3second aerosol spray. Aerosol spray rate was determined to be 0.04L/min. After a 5 minute wait following pretreatment with hydrogenperoxide, the lumen was dried as described above and the lumen was thenplaced in a sterilization chamber and exposed to either a vacuum of 0.5Torr for 15 minutes, or plasma for 15 minutes at 0.5 Torr. The resultsare set forth below in Table 16C.

                  TABLE 16C    ______________________________________    Effects of Aerosol Delivery of H.sub.2 O.sub.2 Solution on    Sporicidal Activity Prior to Either a Vacuum    or a Plasma Treatment in Metal Lumens              Number of Surviving                              Sterility Test Results    Conc. H.sub.2 O.sub.2              Organisms after Aerosol +                                       Aerosol +    (%)       Aerosol Alone   Vacuum   Plasma    ______________________________________    3         7.4 × 10.sup.5                              0/4      0/4    ______________________________________

Flow-through of hydrogen peroxide as either a liquid solution or aerosolcan also be achieved by introducing increased pressure at the deliveryend or decreased pressure at the exit end of the device to be treated.

It is evident from the data in Tables 16A-16C that all three methods ofdelivering hydrogen peroxide solution to the article to be sterilizedprovided for effective sterilization. Thus, it appears that a number ofdifferent methods of delivery can be used, as long as the hydrogenperoxide solution is present in the system prior to exposure to vacuumor plasma.

Finally, the efficacy of pretreatment with hydrogen peroxide prior to asterilization cycle which combines exposure to hydrogen peroxide vapor,vacuum, and plasma was evaluated. The testing was as follows.

EXAMPLE 17

Stainless steel blades were inoculated with 2.5×10⁶ B.stearothermophilus spores. The blades were soaked in 3% hydrogenperoxide solution for either 1 or 5 minutes. The blades were then placedin the expanded center piece of a 3 mm ×50 cm stainless steel lumen. Thelumen was then placed in a sterilization chamber which was evacuated toapproximately 0.5 Torr. The sterilization cycle consisted of 15 minutesof hydrogen peroxide vapor diffusion with a minimum of 6 mg/L hydrogenperoxide, followed by 15 minutes of plasma at 400 watts. Following theplasma treatment, the chamber was vented and the blades tested forsterility. The results are shown below.

                  TABLE 17    ______________________________________    Effects of H.sub.2 O.sub.2 Solution Soak on Sporicidal    Activity in Stainless Steel Lumens Prior to a    Hydrogen Peroxide Vapor and Plasma Cycle                         Sterility Test Results    Conc. H.sub.2 O.sub.2               Soak Time Soak Alone                                   Soak + Cycle    ______________________________________    3%         1 min     4/4       0/4               5 min     4/4       0/4    ______________________________________

Processing the lumens in a hydrogen peroxide vapor and plasma cyclealone left an average of 30 surviving organisms per blade. Pre-treatingthe blades by soaking in 3% hydrogen peroxide solution for 5 minutesalone left an average of 8.2×10⁵ surviving organisms per blade. Thus,under these particular test conditions, a combination of hydrogenperoxide vapor exposure and plasma exposure, which has been found to beeffective for many articles, was ineffective in a diffusion restrictedenvironment. However, by pre-treating the article to be sterilized withdilute hydrogen peroxide solution prior to exposure to hydrogen peroxidevapor and plasma, complete sterilization can be achieved.

While the invention has been described in connection with preferredliquid sterilant solutions containing hydrogen peroxide, it will beappreciated by those having ordinary skill in the art that equivalentsterilization methods can be adapted for other sources of peroxidesterilants. In an alternative embodiment, a sterilant having a vaporpressure lower than that of water or other solvent in which thesterilant may be provided is used. For such sterilants, it is onlyimportant that the vapor pressure be lower than that of the solventwithin the temperature ranges contemplated herein. In yet otherembodiments, a solid source of peroxide sterilant may be utilized. Suchliquid and solid sterilants can be adapted for the techniques describedherein with only minor adjustments made for the differences in vaporpressure between hydrogen peroxide and such other sterilant, as can bereadily determined by those having ordinary skill in the art. As long asthe local vapor pressure at the site of the sterilant is below the vaporpressure of the sterilant, sterilization can be achieved substantiallyas described hereinabove.

Conclusion

Achieving rapid sterilization of lumened devices at low temperaturesusing low concentrations of sterilants has, until now, been exceedinglychallenging. A superior method of sterilization has been discoveredwhich overcomes the problems of the known methods. By pre-treatingarticles to be sterilized or a diffusion-restricted environmentcontaining the articles with a source of peroxide such as an aqueoussolution of hydrogen peroxide prior to exposure to a vacuum, rapidsterilization can be achieved at low temperatures, without damage to thearticles, without leaving toxic residues behind, and without the need toattach special vessels. The method of the present invention isefficient, nonhazardous, and inexpensive as well.

What is claimed is:
 1. A method for sterilizing an article within acontainer in a vacuum chamber, said method comprising:(a) placing saidarticle in said container, wherein said container is in fluidcommunication with the chamber through an exit tube having a length tointernal diameter ratio greater than or equal to 1.1; (b) contacting theinterior of the container with a source of peroxide which releaseshydrogen peroxide vapor, said placing and contacting steps beingperformed in either order; (c) reducing the pressure of the chamber andcontainer below the vapor pressure of hydrogen peroxide; (d) generatinghydrogen peroxide vapor in the container; (e) diffusing said hydrogenperoxide vapor from the container through the exit tube into said vacuumchamber; and (f) exposing said article to said hydrogen peroxide vaporfor a time period sufficient to effect sterilization of said article. 2.The method of claim 1, wherein the exposing step is repeated one or moretimes.
 3. The method of claim 1, wherein the entire method is repeatedone or more times.
 4. The method of claim 1, wherein said contactingstep comprises delivery via one or more methods selected from the groupconsisting of injection, static soak, liquid flow-through, aerosolspray, condensation or physical placement.
 5. The method of claim 1,further comprising the step of exposing said container to a plasmaduring the step of exposing the article to the hydrogen peroxide vapor.6. The method of claim 5, wherein said method is performed within asealed chamber and said plasma is generated within said chamber.
 7. Themethod of claim 1, wherein said exposing step is performed for 60minutes or less.
 8. The method of claim 1, further comprising the stepof heating said container during said exposing step.
 9. The method ofclaim 1, further comprising heating said source of peroxide prior tosaid contacting step.
 10. The method of claim 1, wherein said exposingstep further comprises exposing said container to negative pressurebetween 0 and 100 Torr.
 11. The method of claim 1, wherein said sourceof peroxide comprises a liquid, a solid or condensed vapor.
 12. Themethod of claim 11, wherein said liquid comprises hydrogen peroxide orperacetic acid.
 13. The method of claim 11, wherein said solid comprisesa urea peroxide complex or sodium pyrophosphate peroxide complex or likecomplex.
 14. The method of claim 11, wherein said condensed vaporcomprises hydrogen peroxide or peracetic acid vapor.
 15. The method ofclaim 1, wherein said exit tube has a filter therein.
 16. The method ofclaim 15, wherein said filter prevents entry of bacteria into saidcontainer.
 17. The method of claim 1, wherein said exit tube is at least1.0 cm in length.
 18. The method of claim 1, wherein said exit tube hasan internal diameter of 9 mm or less.
 19. The method of claim 1, whereinsaid exit tube is additionally connected to a valve outside saidcontainer, said valve being in communication with the vacuum chamber.20. A method for sterilizing a lumened article having an exteriorsurface and an interior surface within a container in a vacuum chamber,wherein said container is in fluid communication with the chamberthrough a communication port on the container, said methodcomprising:(a) placing said article in said container, said articlehaving a lumen with a length to internal diameter ratio greater than orequal to 90; (b) connecting the lumen of said article to thecommunication port through a connector; (c) contacting the interior ofthe container with a source of peroxide which releases hydrogen peroxidevapor, said placing, connecting, and contacting steps being performed inany order; (d) reducing the pressure of the chamber and container belowthe vapor pressure of hydrogen peroxide; (e) generating said hydrogenperoxide vapor in the container; (f) diffusing said hydrogen peroxidevapor from the container through the lumen, the connector, thecommunication port and into said vacuum chamber; and (g) exposing saidarticle to said hydrogen peroxide vapor to effect sterilization of theinterior surface and the exterior surface of said article.
 21. Themethod of claim 20, wherein said connector comprises tubing or anadaptor which can be attached to the lumen of said article or anenclosure which contains a portion of said article.
 22. The method ofclaim 20, wherein the container is diffusion restricted.
 23. The methodof claim 22, wherein the diffusion restriction in said container resultsfrom said communication port.
 24. The method of claim 20, wherein saidcommunication port comprises an exit tube.
 25. The method of claim 24,wherein said exit tube is at least 1.0 cm in length.
 26. The method ofclaim 24, wherein said exit tube has an internal diameter of 9 mm orless.
 27. The method of claim 20, wherein said communication portcomprises an air and vapor permeable window.
 28. The method of claim 27,wherein said window is impermeable to microorganisms.
 29. The method ofclaim 20, wherein said container additionally contains one or morearticles not having a lumen.
 30. The method of claim 20, wherein saidcontainer additionally contains an article having a lumen with a lengthto internal diameter ratio less than
 90. 31. The method of claim 20,further comprising the step of exposing said container to a plasmaduring the step of exposing the container to negative pressure.
 32. Themethod of claim 31, wherein said method is performed within a sealedchamber and said plasma is generated in said chamber.
 33. The method ofclaim 20, wherein said container is a rigid container.
 34. The method ofclaim 20, wherein said container is a flexible pouch.